The present invention relates to a novel ErbB-4 ligand, referred to herein as Neuregulin-4 (NRG-4), to polynucleotide sequences encoding said NRG-4, to oligonucleotides and oligonucleotide analogs derived from said polynucleotide sequences, to a display library displaying short peptides derived from said NRG-4, to antibodies recognizing said NRG-4, to peptides or peptide analogs derived from said NRG-4, and to pharmaceutical compositions and methods of employing said peptides or peptide analogs, said oligonucleotides and oligonucleotide analogs, and/or said polynucleotide sequences to up-regulate or down-regulate ErbB-4 receptor activity and to treat or prevent various diseases, conditions and syndromes.
Cell-to-cell signaling is an essential feature of multi-cellular organisms, playing important roles in both the unfolding of developmental diversification as well as mediating the homeostasis of vastly different cell types. A large number of tyrosine kinase growth factor receptors play key roles in this process. Type-1 tyrosine kinase receptors, also known as ErbB/HER proteins, comprise one of the better-characterized families of growth factor receptors, of which the epidermal growth factor receptor (ErbB-1) is the prototype [reviewed in (Burden & Yarden, 1997)]. The ErbB family constitutes four known receptors which dimerize upon ligand stimulation, transducing their signals by subsequent autophosphorylation catalyzed by an intrinsic cytoplasmic tyrosine kinase, and recruiting downstream signaling cascades.
The ErbB receptors are activated by a large number of ligands. Depending upon the activating ligand, most homodimeric and heterodimeric ErbB combinations can be stabilized upon ligand binding (Tzahar et al., 1996), thus allowing a complex, diverse downstream signaling network to arise from these four receptors. The choice of dimerization partners for the different ErbB receptors, however, is not arbitrary.
Spatial and temporal expression of the different ErbB receptors do not always overlap in vivo, thus narrowing the spectrum of possible receptor combinations that an expressed ligand can activate for a given cell type (Erickson et al., 1997; Gassmann et al., 1995; Lee et al., 1995; Pinkas-Kramarski et al., 1997; Riethmacher et al., 1997).
Furthermore, a hierarchical preference for signaling through different ErbB receptor complexes takes place in a ligand-dependent manner. Of these, ErbB-2-containing combinations are often the most potent, exerting prolonged signaling through a number of ligands, likely due to an ErbB-2-mediated. deceleration of ligand dissociation (Karunagaran et al., 1996; Tzahar et al., 1996; Wang et al., 1998).
In contrast to possible homodimer formation of ErbB-1 and ErbB-4, for ErbB-2, which has no known direct ligand, and for ErbB-3, which lacks an intrinsic tyrosine kinase activity (Guy et al., 1994), homodimers either do not form or are inactive.
Heterodimeric ErbB complexes are arguably of importance in vivo. For example, mice defective in genes encoding either NRG-1, or the receptors ErbB-2 or ErbB-4, all result in identical failure of trabeculae formation in the embryonic heart, consistent with the notion that trabeculation requires activation of ErbB-2/ErbB-4 heterodimers by NRG-1 (Gassmann et al., 1995; Lee et al., 1995; Meyer & Birchmeier, 1995).
At the biochemical level, the known ErbB ligands fall into several categories (Riese et al., 1996b). One category, the ErbB-1 ligands, includes EGF, Transforming Growth Factor α (TGFα), Epiregulin, Amphiregulin, Betacellulin and the Heparin-binding EGF (HB-EGF) (Higashiyama et al., 1991; Marquardt et al., 1984; Shing et al., 1993; Shoyab et al., 1989; Toyoda et al., 1995). To different extents, these ErbB-1 binding ligands can also activate other receptors of the ErbB family, and hence may mediate distinct signaling outputs for a given cell type [reviewed in (Tzahar & Yarden, 1998)].
Another category of ErbB ligands comprises the Neuregulin (NRG) family. NRG-1 [also named Neu differentiation factor (NDF), Heregulin, Glial Growth factor, and Acetylcholine Receptor Inducing Activity] was first identified by its ability to indirectly phosphorylate ErbB-2 (Holmes et al., 1992; Peles et al., 1992; Wen et al., 1992). Subsequently, NRG-1 was found to directly bind ErbB-3 and ErbB-4 and to sequester ErbB-2 by receptor dimerization (Peles et al., 1993; Plowman et al., 1993; Sliwkowski et al., 1994; Tzahar et al., 1994). Multiple isoforms of NRG-1 exist which amongst other roles, permit heterogeneous binding affinities to different ErbB complexes (Tzahar et al., 1994). The NRG family now includes also two isoforms of NRG-2 (Busfield et al., 1997; Carraway et al., 1997; Chang et al., 1997; Higashiyama et al., 1997), of which the alpha isoform is a pan-ErbB ligand (Pinkas-Kramarski et al., 1998), and NRG-3, a ligand of ErbB-4 (Zhang et al., 1997).
The multiplicity of genes encoding ErbB-1 ligands, contrasting with the small number of known genes encoding ligands for ErbB-3 or ErbB-4 (namely: NRGs), led the inventors of the present invention to believe in the existence of additional NRG genes in the genome of mammals.
A fourth Neuregulin, denoted NRG-4, which acts through the ErbB-4 receptor tyrosine kinase is reported herein. In addition to its novel structure, this growth factor displays a pattern of expression different from other EGF-like molecules.